Tag: brain-derived neurotropic factors

Jan Nolta and her colleagues at the Stem Cell Program and Institute for Regenerative Cures at UC Davis have published a remarkable paper in the journal Molecular Therapy regarding Huntington’s disease and a potential stem cell-based strategy to delay the ravages of this disease.

Huntington’s disease (HD) is an inherited neurodegenerative disease. It is inherited as an autosomal dominant disease, which means that someone need only inherit one copy of the disease-causing allele of the HTT gene to have this disease. HD is characterized by progressive cell death in the brain, particularly in a portion of the brain known as the striatum and by widespread brain atrophy.

The portion of the brain known as the striatum lies underneath the surface of the forebrain (subcortical) and it receives neural inputs from the cerebral cortex and is the primary source of neural inputs to the basal ganglia system. The basal ganglia system (BGS) is located underneath the surface of the brain but even deeper within the cerebral hemispheres. The BGS is part of the corpus striatum, it consists of the subthalamic nucleus and the substantia nigra. The BGS help with voluntary motor control, procedural learning relating to routine behaviors. otherwise known as “habits,” eye movements, and cognitive, and emotional functions. The ventral striatum is very important in addiction because it is the reward center on consists of the nucleus accumbens, olfactory tubercle, and islands of Calleja.

This is a transverse section of the striatum from a structural MR image. The striatum, in red, includes the caudate nucleus (top), the putamen (right), and, when including the term ‘corpus’ striatum, the globus pallidus (lower left).

HD takes its largest toll on the striatum, which affects voluntary movement, routine behaviors, and personality. Disturbances of both involuntary and voluntary movements occur in individuals with HD. Chorea, an involuntary movement disorder consisting of nonrepetitive, non-periodic jerking of limbs, face, or trunk, is the major sign of the disease. Chorea is present in more than 90% of individuals, increasing during the first ten years. The choreic movements are continuously present during waking hours, cannot be suppressed voluntarily, and are worsened by stress. HD patients show impaired voluntary motor function early on and show a clumsiness in common daily activities.

Animal models of HD used in the past have injected molecules into the brain that kill off striatal cells and mimic at least some of the characteristics of HD in laboratory animals. Unfortunately, such a model system is fat too clean, since implanted cells tend to survive perfectly well. However the brains of HD patients are like unto a toxic waste dumps and implanted cells are quickly killed off. Therefore, a better animal model system was required, and it came in the form of R6/2 and YAC128 mice. R6/2 mice have a part of the human HTT gene that has 150 CAG triplets, and show the characteristic cell death in the striatum and behavioral deficits. The only problem with this mouse strain is that the neurodegenerative decline is very rapid rather than slow and progressive. YAC128 mice have a full-length copy of the HTT gene and show a slower, more progressive neurological decline that more closely approximates the human clinical condition.

In this paper from the Nolta laboratory, they used a growth factor that is known to decrease precipitously in HD brains; a growth factor called Brain-Derived Neurotrophic Factor (BDNF). BDNF is known to mediate the survival and function of striatal neurons and the reduction of BDNF in the brains of HD patients correlates with the onset of symptoms and the greater the reduction in BDNF, the greater the severity of the disease (see Her LS & Goldstein LS, J Neurosci 2008; 28, 13662-13676).

However injecting BDNF into the brain is problematic, since the protein has a very short half-life. Delivering the growth factor by means of genetically engineered viruses shows promise, but most of the viral vectors used in such experiments are recognized by the immune system as foreign invaders. Therefore, Nolta and her colleagues decided to genetically engineer mesenchymal stem cells (MSCs) to overexpress BDNF and implant these cells into the brains of R6/2 and YAC128 mice.

Nolta and her coworkers actually tested human MSCs in HD model mice. After completing all the necessary control experiments to ensure that their isolated and engineered MSCs were secreting BDNF, Nolta and others implanted them into the brains of R6/2 and YAC128 mice.

HD mice show greater anxiety, which is manifested in a so-called “open field assay” by not remaining the center of the field. The control HD mice did not stay long in the center of the open field, but the normal mice did. The MSC-BDNF-implanted mice spend far more time in the center of the field. Mind you, not as much as wild-type mice, but significantly more than their HD counterparts.

Next the volume of the striata of these mice were determined and compared to the normal mice. While all the HD mice showed shrinking of the striatum, the MSC-NDNF-implanted YAC128 mice show significantly less shrinking of the striatum. Then the degree of neurogenesis (formation of new neurons) was measured in normal, HD, HD + implanted MSCs, and HS + MSC-BDNF mice. This experiment measures the degree of healing that is occurring in the brain. The brain from HD + MSC and HD + MSC-BDNF mice showed significantly more new brain cell growth. This is probably the reason for the delayed onset of symptoms and the delayed shrinking of the striatum.

Finally, Nolta and others measured the lifespans of the R6/2 mice and compared them with R6/2 mice that had been implanted with MSCs-BDNF. Animals transplanted with the MSCs that made the most BDNF lived 15% longer than the nontreated R6/2 mice.

MSCs have been shown in several experiments to promote neuronal growth, decrease cell death and decrease inflammation through the secretion of trophic factors. MSCs can modify the toxic environment that is part of the brain of an HD patient and help damaged tissue out by inducing neural regeneration and protection (see Crigler L, et al., Experimental neurology, 198; 2–6, 54-64; Kassis I, et al., Archives of Neurology 65; 2008: 753-761).

The downside of using MSCs that they will only survive in the brain for a few months. However, several studies have shown that the benefits of modified MSC implantation persist after the MSCs are gone, since the neural reconstruction wrought by the secreted BDNF stay after the MSCs have died off (see Arregui L, et al., Cell Mol Neurobiol 31; 2011: 1229-1243 and many others).

At best this treatment would delay the ravages of HD, but delaying this disease might very well be the first step towards a cure. Hopefully, clinical trials will not be fat behind.

Lewy bodies are aggregations of misfolded proteins in nerve cells that can kill them off and cause dementia. When Lewy bodies form in neurons, they can cause “dementia with Lewy bodies” or DLB. After Alzheimer’s disease, DLB is the second-most common type of age-related dementia, and it afflicted the beloved comedian Robin Williams, who took his own life earlier this year.

Scientists at the Sue and Bill Gross Stem Cell Research Center and the Institute for Memory Impairments and Neurological Disorders at UC Irvine have examined the ability of transplanted neural stem cells to ameliorate the symptoms of DLB is an animal model system.

Particular strains of laboratory mice have been genetically engineered to form Lewy bodies in their brains and show some of the symptoms of DLB. Natalie Goldberg and her colleagues used neural stem cells to treat some of these mice in order to determine if these cells could decrease the pathological consequences of DLB.

Transplantation of neural stem cells into the brains of these DLB mice resulted in increases in cognitive and motor function. A battery of tests established this. For example, the Rotarod test places the mouse on a rod that is then rotated at a specific speed. Normal mice can move around the rod and keep from falling off, but mice with motor or balance problems will fall off the rod prematurely. Cognitive tests included Novel Object Recognition (NOR) and Novel Place Recognition (NPR) tests, which are low-stress tasks that quantify the proportion of time spent examining a novel object and provide data on memory. In these tests, the mice that received the neural stem cell transplantation did significantly better than their non-treated siblings.

Goldberg and his team then asked how these cells improved the cognitive and motor function of the DLB mice. It turns out that neutral stem cells secrete respectable amounts of brain-derived neurotrophic factor (BDNF). Goldberg suspected that this growth factor was a major contributor to the healing capabilities of neural stem cells. Therefore, Goldberg’s team engineered neural stem cells that could not make BDNF and injected those directly into the brains of DLB mice. These mutant neural stem cells were incapable of improving the cognitive or motor function of these mice.

To further test her hypothesis, Goldberg then engineered a virus that would infect neurons and overexpress BDNF and used that to treat her DLB mice. Interestingly, the BDNF-expressing virus did a pretty good job at restoring motor functions in these DLB mice, but did not restore the cognitive functions.

Thus, while the secretion of BDNF by neural stem cells is important for their restorative capacities, but it is only part of the means they use to heal affected brains. Goldberg and her coworkers showed that the transplanted neural stem cells did not improve the pathology of the brains, they did preserve neural pathways that use the neurotransmitters dopamine and glutamate.

The neural stem cells used in these experiments were mouse neural stem cells. Before work like this can advance to human clinical trials, human neural stem cells must be tested. Since other neurodegenerative diseases like Parkinson’s disease also result from Lewy body formation in specific cells, neural stem cell treatments might prove beneficial for patients with much diseases.

This work was published in Stem Cell Reports October 2015 DOI: 10.1016/j.stemcr.2015.09.008.

Vicki Wheelock at the UC Davis Medical Center has registered clinical trial number NCT01937923, which is otherwise known as “PRE-CELL.” This clinical trial will use various imaging techniques, laboratory tests, and clinical evaluations of Huntington’s disease (HD) patients to map the disease progression over 12-18 months. This trial will then hopefully identify candidates for a new trial in which these patients will be implanted with mesenchymal stem cells that secrete nerve growth factors. This represents one of the first clinical trials to examine the use of mesenchymal stem cells in the treatment of HD

The rationale for this study comes from a 2012 study in mice. Ofer Sadan, Eldad Melamed, and Daniel Offen from the Rabin Medical Center in Tel Aviv University, Israel, used R6/2 mice to test the efficacy of nerve growth factor-secreting mesenchymal stem cells isolated from bone marrow . In this paper, Sadan and others isolated mesenchymal stem cells from the bone marrow of healthy human volunteers and mice and then cultured them in special growth media that induces these cells to secrete special nerve growth factors. These so-called NTF+ cells were then transplanted into the striatum of R6/2 mice.

R6/2 mice express part of the human HTT gene; specifically the part that causes HD. Since HD is an inherited disease, there is a specific gene responsible for the vast majority of HD cases, and that gene is the human HTT gene, which encodes the Huntington protein. The function of the Huntington protein is uncertain, but it is found at high levels in neurons, even though it is found in other tissues as well, and dysfunctional Huntington protein affects neuron health.

The HTT gene in HD patients contains the insertion of extra copies of the CAG triplet. The more CAG triplets are inserted into the HTT gene, the more severe the HD caused by the mutation. The hitch is that normal copies of the HTT gene has multiple copies of this CAG repeat. CAG encodes the amino acid glutamine, and Huntington contains a stretch of glutamine residues that seem to allow the protein to interact with other proteins found in neurons. When this glutamine stretch becomes too long, the protein is toxic and it begins to kill the cells. How long is too long? Research has pretty clearly shown that people whose HTT genes contain less than 28 CAG virtually never develop HD. People with between 28–35 CAG repeats, are usually unaffected, but their children are at increased risk of developing HD. People whose HTT genes contain 36–40 CAG repeats may or may not show HD symptoms, and those who have over 40 copies almost always are afflicted with HD.

Now, back to R6/2 mice. These animals contain a part of the human HTT gene that has 150 CAG triplets. These mice show the characteristic cell death in the striatum and have behavioral deficits. In short R6/2 mice are pretty good model systems to study HD.

Sadan and others implanted MSCs that had been conditioned in culture to express high levels of nerve growth factors. Then these cells were transplanted into the striatum of R6/2 mice. R6/2 mice were also injected with buffer as a control.

The results showed that injections of NTF+ MSCs before the onset of symptoms did little good. The mice still showed cell death in the brains and behavioral deficits. However, NTF+ MSCs injected later (6.5 weeks), resulted in temporary improvement in the ability of the R6/2 mice to move and these cells also extended their life span. These results were published in the journal PLoS Currents (2012 Jul 10;4:e4f7f6dc013d4e).

Other work, also by Sadan and others, showed that injected MSCs tended to migrate to the damaged areas. When the injected cells were labeled with iron particles, they could be robustly observed with MRIs, and MRIs clearly showed that the injected cells migrated to the damaged areas in the brain (Stem Cells 2008; 26(10):2542-51). Another paper by Sadan and others also demonstrated that the striatum of NTF+ MSC-injected mice show less cell death than control mice (Sadan, et al. Exp Neurol. 2012; 234(2): 417-27). Other workers have also shown that implanted MSCs can provide improve symptoms in R6/2 mice and that they primary means by which they do this is by the secretion of nerve growth factors (Lee ST, et al. Ann Neurol 2009; 66(5): 671-81).

Thus, there is ample reason to suspect the PRECELL trial may lead to a stem cell-based clinical trial that will yield valuable clinical information. The animal data shows definite value in using preconditioned MSCs as a treatment for HD, and if the proper patients are identified by the PRE-CELL trials, then hopefully it will lead to a “CELL” trial in which HD patients are treated with NTF+ MSCs.

Mind you, this treatment will only delay HD at best and buy them time. Such treatments will not cure them. The NTF+ MSCs survive for a finite period of time in the hostile environment of the striatum of the HD patient, and the relief they will provide will be temporary. MSCs do not differentiate into neurons in this case, and they do not replace dead neurons, but they only help spare living neurons from suffering the same fate.

There is an MSC cell line that does make neurons, and if this cell line were used in combination with NTF+ MSCs, then perhaps neural replacement could be a possibility. Also neural precursor cells could be used in combination with NTF+ MSCs to increase their survival. Even then, as long as diseased neurons are producing toxic products, until gene therapy is perfected to the point that the actual genetic lesion in the striatal neurons is fixed, the deterioration of the striatum is inevitable. However, treatments like this could, potentially, delay this deterioration. This clinical trial should give us more information on exactly that question.

Two more points are worth mentioning. When fetal striatal grafts were implanted into the brains of HD patients, the grafts underwent disease-like degeneration, and actually made the patients worse (see Cicchetti et al. PNAS 2009; 106(30): 12483-8 and Cicchetti F, et al. Brain 2011; 134(pt 3): 641-52). Straight fetal implants do not seem to work. Please let’s put the kibosh on these gruesome experiments. Secondly, when neuronal precursor cells differentiated from human embryonic stem cells were implanted into HD rodents, the implanted cells formed some neurons and improved behavior to some extent, but non-neuronal differentiation remained a problem (Song J, et al., Neurosci Lett 2007; 423(1): 58-61). Having non-brain cells in your brain is a significant safety problem. Thus, embryonic stem cell-derived neuronal precursor cells do not seem to be the best bet to date either. So, this present clinical trial seems to be making the most of what is presently safely available.

However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.

Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.

A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).

The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.

All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups. Thus injecting more cells does not lead to greater numbers of surviving neural stem cells. This makes sense, since the damaged spinal cord in very inhospitable place for transplanted cells.

However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.

Human Astrocytes

Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”

Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.